BCH 331 (METHODS IN BIOCHEMISTRY) NOTES 2024

SEPARATION TECHNIQUES
INTRODUCTION

Great strides have been made over the centuries, to biochemically analyse the various body
fluids (especially urine, blood and cerebrospinal fluid) and tissues. Various methods are used
for separation, identification and purification of biological samples, using modern
instruments. Biochemistry offers access to techniques invaluable in determining purity,
stability, size, conformation and finding the characteristics of biomolecules (proteins, DNA,
low molecular weight ligands and assemblies). Many of the strategies available are “in
solution”; free of interactions with additional matrices, surfaces and labelling, and therefore
better suited to understanding biological processes. Among some of the methods used are the
following:

SPECTROSCOPY
Spectroscopy is the study of the absorption and emission of light and other radiation by
matter. It involves the splitting of light (or more precisely electromagnetic radiation) into its
constituent wavelengths (a spectrum), which is done in much the same way as a prism splits
light into a rainbow of colours. Spectrophotometry on the other hand, is the method used to
measure the absorption of light in a chemical substance.

Spectrophotometry
Spectrophotometry is the quantitative measurement of the interaction of ultraviolet (UV),
visible, and infrared (IR) radiation with a material and has an impact on a wide field of
science and technology. The nature of this interaction depends upon the physical properties of
the material, for example, transparent or opaque, smooth or rough, pure or contaminated, and
thin or thick. Thus, spectrophotometric measurements can be used to quantify these important
physical properties of the material. The choices of spectrophotometric measurements include
spectral reflectance, transmittance, absorptance, emittance, scattering and fluorescence; and
these can be classified as the phenomenal optical properties of the material being
investigated.

Principle: When a beam of incident light of intensity, Io passes through a solution, a part of it
is reflected (Ir), a part absorbed (Ia) and rest transmitted (It), i.e., Io = Ir + Ia + It In colorimetric
methods, Ir is eliminated because the measurement of Io and It will be sufficient to determine

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Ia. For this purpose, the amount of light reflected (or I r) is kept constant by using cells that
have identical properties. Io and It are then measured. The mathematical relationship between
the amount of light absorbed and the concentration of a substance can be shown by the
following two fundamental laws, on which the spectrophotometry is based.

Distribution of incident light passing through a solution contained in a cell

A. Lambert's law (Bouguer's law): This law states that the amount of light absorbed is
directly proportional to the length or thickness of the solution under analysis.

Thus

where A = absorbancy,

as = absorbancy index characteristic for the solution

b = length or thickness of the medium

B. Beer's law: This law states that the amount of light absorbed is directly proportional to the
concentration of the solute in solution. Thus:

where c = concentration of solute in solution

The combined Beer-Lambert Law then becomes:

If b is kept constant by employing a standard cell or cuvette, the above formula reduces to:

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The absorbency index, as is defined as:

where C = concentration of absorbing material in g/litre L =distance in cm travelled by the

light in solution.

If one wishes to express the light absorption in terms of the molar concentration of the
absorbing material, the molar absorbency index, am will be equal to:

where M = molecular weight of the absorbing material

Spectrophotometer: is an instrument is an instrument that measures the amount of light

absorbed by a sample. It makes use of techniques that are mostly used to measure the
concentration of solutes in solution by measuring the amount of the light that is absorbed by
the solution in a cuvette placed in the spectrophotometer. It was invented by the scientist
Arnold J. Beckman and his colleagues at the National Technologies Laboratory (NTL), where
they invented the Beckman DU spectrophotometer in 1940.

The technique used by the spectrophotometer is one that measures light intensity as a
function of wavelength. It does this by diffracting the light beam into a spectrum of
wavelengths, detecting the intensities with a charge-coupled device, and displaying the results
as a graph on the detector and then on the display device.

1. In the spectrophotometer, a prism (or) grating is used to split the incident beam into
different wavelengths.

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 2. By suitable mechanisms, waves of specific wavelengths can be manipulated to fall on
the test solution. The range of the wavelengths of the incident light can be as low as 1
to 2nm.

3. The spectrophotometer is useful for measuring the absorption spectrum of a

compound, that is, the absorption of light by a solution at each wavelength.

The essential components of spectrophotometer instrumentation include:

1. A table and cheap radiant energy source

 Materials that can be excited to high energy states by a high voltage electric discharge
(or) by electrical heating serve as excellent radiant energy sources.

2. A monochromator, to break the polychromatic radiation into component wavelength

(or) bands of wavelengths.

 A monochromator resolves polychromatic radiation into its individual wavelengths

and isolates these wavelengths into very narrow bands. The can be classified into:

Prisms

 A prism disperses polychromatic light from the source into its constituent
wavelengths by virtue of its ability to reflect different wavelengths to a different
extent

 Two types of Prisms are usually employed in commercial instruments. Namely,

600 cornu quartz prism and 300 Littrow Prism.

Grating

 Gratings are often used in the monochromators of spectrophotometers operating

ultraviolet, visible and infrared regions.

3. Transport vessels (cuvettes), to hold the sample

 Samples to be studied in the ultraviolet (or) visible region are usually glasses (or)
solutions and are put in cells known as “CUVETTES”.

 Cuvettes meant for the visible region are made up of either ordinary glass (or)
sometimes Quartz.

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 4. A Photosensitive detector and an associated readout system

 Most detectors depend on the photoelectric effect. The current is then proportional to
the light intensity and therefore a measure of it.

 Radiation detectors generate electronic signals which are proportional to the

transmitter light.

 These signals need to be translated into a form that is easy to interpret.

 This is accomplished by using amplifiers, Ammeters, Potentiometers and

Potentiometric recorders.

Operational Procedure of a Spectrophotometer

1. Preparation

o Warm-up the Instrument: Turn on the spectrophotometer and allow it to

warm up for a few minutes. This ensures the light source stabilizes for
accurate readings.

o Select the Wavelength: Choose the appropriate wavelength corresponding to

the absorption peak of the analyte. Refer to literature or calibration data for
guidance.

2. Calibration (Blanking)

o Prepare the Blank Solution: Use a solvent or medium identical to the one in
which the sample is dissolved, but without the analyte.

o Insert the Blank: Pour the blank solution into a clean cuvette, ensuring it's
free of bubbles, fingerprints, or scratches.

o Set the Baseline: Place the blank in the spectrophotometer's sample holder
and adjust the instrument to set the absorbance to zero (or transmittance to
100%).

3. Sample Preparation

o Prepare the Sample: Ensure the analyte is dissolved or suspended uniformly.

Dilute if necessary to stay within the instrument's detection range.

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 o Fill the Cuvette: Pour the prepared sample into a clean cuvette. Handle the
cuvette by its frosted sides to avoid smudges on the optical path.

4. Measurement

o Insert the Sample: Place the cuvette containing the sample in the
spectrophotometer's sample holder, aligning it properly with the light beam.

o Record the Absorbance or Transmittance: Close the lid (if applicable) and
press the measurement button. The spectrophotometer will display the
absorbance (or transmittance) at the selected wavelength.

o Repeat the measurement for additional samples as needed.

5. Data Analysis

o Plot a Calibration Curve: If quantitative analysis is required, prepare a

calibration curve using standard solutions of known concentrations.

o Determine the Concentration: Use the sample's absorbance value and the
calibration curve (or Beer's Law equation) to calculate the concentration of the
analyte.

6. Post-Use Maintenance

o Clean the Cuvettes: Wash the cuvettes thoroughly with distilled water or an
appropriate solvent to prevent contamination.

o Turn Off the Instrument: Power down the spectrophotometer and cover it to
protect it from dust when not in use.

Applications

Some of the major applications of spectrophotometers include the following:

 Detection of concentration of substances

 Detection of impurities

 Structure elucidation of organic compounds

 Monitoring dissolved oxygen content in freshwater and marine ecosystems

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  Characterization of proteins

 Detection of functional groups

 Respiratory gas analysis in hospitals

 Molecular weight determination of compounds

 The visible and UV spectrophotometer may be used to identify classes of compounds

in both the pure state and in biological preparations.

MASS SPECTROSCOPY

Mass spectrometry (MS) is a technique wherein chemical substances are identified in

biological and non-biological material. It uses electric and magnetic fields to separate
gaseous ions based on their mass-to-charge ratios. The MS is a technique that uses a mass
spectrometer and mass spectrograph as instruments that work by using electric and magnetic
fields, and photographic techniques or non-electric means, respectively. MS is used to
identify the different gases present in the atmosphere. Mass spectrometry (MS) is a sensitive,
quantitative, and analytical technique which involves the separation of gaseous ions from the
liquid or solid-state of the samples. After conversion into a gaseous state, these are separated
based on their mobility in an electric and magnetic field. The detected ions or separated ions
are analyzed in a mass spectrum. A mass spectrum is a pattern showing the distribution of
ions by mass in a sample. When a pure substance is present in the sample, MS shows a high
m/z (mass/charge) value and the highest abundance in the mass spectrum.

Principle: The principle involved in mass spectrometry is the formation of several ions from
the sample. Further, these ions are separated according to their mass to charge ratio, which is
also expressed as m/z and then taking a record of the relative abundance of each ion.

Mass Spectrometry (MS) Overview

Mass spectrometry is an analytical technique used to measure the mass-to-charge ratio

(m/z) of ions. It is used to determine the molecular mass of compounds, identify chemical
structures, and quantify the amount of a substance in a sample. The process involves ionizing
molecules and then measuring the abundance of ions as they pass through a magnetic or
electric field. Mass spectrometry is an invaluable tool in many scientific fields, offering
precise, sensitive, and rapid analysis of the chemical composition of substances.

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Understanding the principles behind MS, including ionization, fragmentation, and detection,
is key to making the most of this technique for both qualitative and quantitative analyses.

Basic Principles of Mass Spectrometry

1. Ionization: The sample is first ionized to produce charged particles (ions). This step
is crucial because mass spectrometers only detect ions, not neutral molecules.

Common Ionization Techniques:

o Electron Impact (EI): A high-energy electron beam knocks electrons off the
sample molecules, creating positively charged ions (usually in the gas phase).

o Electrospray Ionization (ESI): The sample is dissolved in a solvent and

sprayed through a charged needle, producing ions directly from the solution.

o Matrix-Assisted Laser Desorption/Ionization (MALDI): A laser pulse hits

the sample, causing it to ionize.

o Chemical Ionization (CI): A reagent gas is used to ionize the sample

molecules via proton transfer or other reactions.

2. Acceleration: Once the ions are formed, they are accelerated by an electric field. This
gives the ions the same kinetic energy, but their velocity will vary depending on their
mass-to-charge ratio (m/z).

3. Separation: The accelerated ions pass through a magnetic or electric field that
separates them based on their m/z ratio. Lighter ions bend more than heavier ones,
and ions with different charges will have different responses.

Types of Mass Spectrometers:

o Quadrupole Mass Spectrometer: Uses four rods to create an oscillating

electric field that selectively stabilizes ions of specific m/z ratios.

o Time-of-Flight (TOF) Mass Spectrometer: Ions are accelerated and then

drift through a field-free region, with heavier ions taking longer to reach the
detector.

o Ion Trap Mass Spectrometer: Ions are trapped in an electric field and then
selectively ejected to the detector.
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 o Orbitrap Mass Spectrometer: Uses an electrostatic field to trap ions in orbit
around a central electrode, separating ions based on their m/z ratio.

4. Detection: The separated ions are detected by a detector (such as an electron

multiplier), which converts the number of ions into a signal, creating a mass spectrum.

Interpretation of Mass Spectrum

A mass spectrum consists of a plot with:

 X-axis: m/z (mass-to-charge ratio)

 Y-axis: Ion intensity (abundance of ions)

Key Features:

1. Molecular Ion Peak (M+): The peak corresponding to the unfragmented molecule. It
represents the molecular mass of the compound.

2. Base Peak: The tallest peak in the spectrum. It represents the most abundant ion, not
necessarily the molecular ion.

3. Fragmentation Pattern: In some cases, ions break apart into smaller fragments
during ionization. The pattern of fragmentation can be used to infer the structure of
the molecule.

4. Isotope Patterns: Many elements have isotopes (e.g., carbon-12 and carbon-13),
which result in small peaks at higher m/z ratios, separated by a regular interval (e.g., 1
m/z unit for carbon isotopes).

Types of Mass Spectrometry Techniques

1. High-Resolution Mass Spectrometry (HRMS): Offers precise measurement of the

m/z ratio, allowing for accurate determination of molecular formulas by resolving
very small differences in mass.

2. Tandem Mass Spectrometry (MS/MS): Involves multiple stages of mass

spectrometry. The first stage (MS1) separates ions, which are then fragmented in a
collision cell. The second stage (MS2) analyzes the fragments, providing more
structural information. MS/MS is particularly useful for:

o Structural elucidation
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 o Peptide sequencing in proteomics

o Analyzing complex mixtures

3. Triple Quadrupole Mass Spectrometry (QQQ): A type of tandem mass

spectrometer with three quadrupoles that are used for precise, quantitative analysis
and targeted fragmentation studies.

Applications of Mass Spectrometry

1. Molecular Identification and Structure Elucidation:

o Identifying unknown compounds by measuring the m/z ratios of molecular

and fragment ions.

o Determining the molecular structure by analyzing the fragmentation patterns.

2. Proteomics:

o Characterizing proteins and peptides through peptide mass fingerprinting.

o Quantifying proteins and identifying post-translational modifications.

3. Metabolomics:

o Studying metabolites in biological samples to understand metabolic processes.

o Monitoring biomarkers for disease diagnosis.

4. Environmental and Forensic Analysis:

o Detecting pollutants, drugs, and toxins in environmental or forensic samples.

5. Pharmaceutical and Drug Testing:

o Analyzing drugs for purity, formulation, and pharmacokinetics.

o Monitoring drug metabolites in clinical trials.

6. Petrochemical and Food Industry:

o Analyzing complex mixtures of hydrocarbons in petroleum products.

o Identifying contaminants and additives in food products.

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ELECTRON SPIN RESONANCE (ESR) / ELECTRON PARAMAGNETIC
RESONANCE (EPR)

Electron Spin Resonance (ESR), also called Electron Paramagnetic Resonance (EPR), is
a spectroscopic technique that measures the interaction of unpaired electrons with an external
magnetic field. It is a powerful tool for studying paramagnetic species (molecules or atoms
with unpaired electrons) such as free radicals, metal complexes, and certain types of
transition metal ions. It is a powerful tool for studying the magnetic properties of unpaired
electrons in a wide range of materials, including free radicals, metal ions, and complex
organic and inorganic systems. By analyzing the ESR spectra, scientists can gain insights into
molecular structure, reactivity and dynamics at a level of detail not accessible through other
techniques.

Key Principles of ESR

The phenomenon of electron spin resonance (ESR) is based on the fact that an electron is a
charged particle. It spins around its axis and this causes it to act like a tiny bar magnet. When
a molecule or compound with an unpaired electron is placed in a strong magnetic field The
spin of the unpaired electron can align in two different ways creating two spin states:

ms = ± ½.

The alignment can either be along the direction (parallel) to the magnetic field which
corresponds to the lower energy state ms = – ½ Opposite (antiparallel) to the direction of the
applied magnetic field ms = + ½

The two alignments have different energies and this difference in energy lifts the degeneracy
of the electron spin states. The energy difference is given by:

∆ E = E+ – E- = hv = gmßB

Where:

h = Planck’s constant (6.626 x 10-34 J s-1)

v = the frequency of radiation

ß = Bohr magneton (9.274 x 10-24 J T-1) B = strength of the magnetic field in Tesla

g = the g-factor which is a unit less measurement of the intrinsic magnetic moment of the
electron, and its value for a free electron is 2.0023.
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Basic ESR Experimental Setup

1. Magnetic Field:

o The magnetic field is applied along a specific direction, typically in the x, y, or

z axis, depending on the experimental setup.

o The magnetic field can be varied in order to detect different resonance

conditions.

2. Microwave Radiation:

o A microwave source is used to provide the energy needed to excite the

electron spins between the different energy levels.

3. Sample Holder:

o The sample, often in a liquid or solid state, is placed in the microwave cavity
(in the case of a cavity-based ESR spectrometer) or near the microwave source
and detector.

4. Detection:

o The resonant absorption of microwave radiation by the unpaired electron(s) is

detected as a change in the amplitude or intensity of the microwave signal.

o The output is usually presented as a spectrum of microwave absorption vs.

magnetic field strength (B0).

ESR Spectral Features

1. The ESR Spectrum:

o The spectrum consists of one or more peaks corresponding to the transitions

between the spin states of the electron(s).

o The position of the peaks gives information about the g-factor and the
magnetic environment of the paramagnetic species.

2. Hyperfine Splitting:

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 o If the unpaired electron is near a nucleus with a non-zero magnetic moment
(e.g., hydrogen or nitrogen), the interaction between the electron’s magnetic
moment and the nucleus’s magnetic moment causes additional splitting of the
spectral lines.

o This results in hyperfine splitting, which appears as a series of closely spaced

peaks around the main resonance line.

o The number and pattern of these splittings depend on the number and type of
nearby nuclei (such as hydrogen, nitrogen, etc.).

3. g-Factor:

o The value of the g-factor provides critical information about the type of
paramagnetic species being analyzed.

 For a free electron, the g-factor is approximately 2.0023.

 For transition metal ions, the g-factor can vary depending on the
electronic configuration and the bonding environment.

4. Line Shape:

o The line shape of an ESR signal is influenced by various factors, including:

 Relaxation times (T1 and T2), which describe how quickly the system
returns to equilibrium.

 Dynamical interactions of the unpaired electrons, such as interactions

with other molecules or with the lattice in a solid.

Factors Affecting ESR Spectra

1. Electron Environment:

o The presence of nearby nuclei, such as hydrogen or nitrogen, causes hyperfine

splitting.

o The chemical environment and bonding (e.g., organic vs. inorganic species)
also influence the ESR spectrum.

2. Temperature:

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 o ESR spectra can change with temperature. At lower temperatures, the
linewidth may narrow, and certain spin states may become more populated.

o The spin-lattice relaxation time (T1) also depends on temperature.

3. Solvent and Matrix Effects:

o In solution, the type of solvent can affect the g-factor and linewidth. In a solid,
the matrix can influence the interactions between paramagnetic centers.

Applications of ESR

1. Free Radical Detection:

o ESR is particularly useful for detecting free radicals, which are molecules or
atoms with unpaired electrons. This includes applications in chemistry,
biochemistry, and materials science.

2. Characterizing Transition Metal Complexes:

o ESR is used to study transition metal ions (e.g., Cu2+Cu^{2+}Cu2+,

Fe3+Fe^{3+}Fe3+) and their complexes, providing information on their
electronic structure, coordination environment, and bonding.

3. Study of Paramagnetic Centers:

o It helps in understanding the behavior of paramagnetic centers in various

materials, such as in catalysts, semiconductors, and magnetic materials.

4. Spin Labeling in Biological Systems:

o ESR is used in spin labeling to study the dynamics and structure of

biomolecules, particularly proteins and nucleic acids, by attaching
paramagnetic probes to the molecules.

5. Reaction Mechanisms:

o ESR can be used to investigate the mechanisms of reactions involving free

radicals or paramagnetic intermediates, which is especially important in fields
like organic chemistry and polymer science.

6. Toxicology:

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 o ESR can monitor the presence of free radicals generated by various
environmental factors (such as radiation or pollutants), which are implicated in
processes like aging, cancer, and neurological diseases.

Advantages of ESR

 Non-destructive: Unlike some techniques, ESR does not destroy the sample.

 High sensitivity: It is highly sensitive to paramagnetic species and can detect even
small amounts of free radicals or metal ions.

 Detailed information: ESR provides detailed information about the electronic

structure and environment of the unpaired electron.

Limitations of ESR

 Requires paramagnetic species: Only species with unpaired electrons are detectable,
so ESR cannot be used to analyze diamagnetic species (most stable compounds).

 Requires specialized equipment: ESR instrumentation can be expensive and

requires a skilled operator.

 Line broadening: In complex samples, overlapping signals or broadening of lines can

complicate spectral interpretation.

Key Notes:

 Always handle cuvettes with care and avoid introducing contaminants.

 Ensure the spectrophotometer is in a stable environment to minimize fluctuations due

to temperature or vibrations.

 Regularly calibrate the instrument and perform routine maintenance to ensure

consistent performance.

NUCLEAR MAGNETIC RESONANCE (NMR)

NMR is a spectroscopic technique used to determine the structure, dynamics, and

environment of molecules based on the magnetic properties of atomic nuclei. It uses a
magnetic field to study the atomic nuclei of a sample, and is used to determine the sample's
structure, purity and content. NMR works by disturbing atomic nuclei in a strong magnetic

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field with a weak oscillating magnetic field. The nuclei respond by producing an
electromagnetic signal with a frequency that's characteristic of the magnetic field.

Principle:

The principle of NMR usually involves three sequential steps:

 The alignment (polarization) of the magnetic nuclear spins in an applied, constant

magnetic field B0.

 The perturbation of this alignment of the nuclear spins by a weak oscillating magnetic
field, usually referred to as a radio frequency (RF) pulse. The oscillation frequency
required for significant perturbation is dependent upon the static magnetic field (B0)
and the nuclei of observation.

 The detection of the NMR signal during or after the RF pulse, due to the voltage
induced in a detection coil by precession of the nuclear spins around B0. After an RF
pulse, precession usually occurs with the nuclei's Larmor frequency and, in itself,
does not involve transitions between spin states or energy levels.

The two magnetic fields are usually chosen to be perpendicular to each other as this
maximizes the NMR signal strength. The frequencies of the time-signal response by the total
magnetization (M) of the nuclear spins are analyzed in NMR spectroscopy and magnetic
resonance imaging. Both use applied magnetic fields (B0) of great strength, usually produced
by large currents in superconducting coils, in order to achieve dispersion of response
frequencies and of very high homogeneity and stability in order to deliver spectral resolution,
the details of which are described by chemical shifts, the Zeeman effect, and Knight shifts (in
metals). The information provided by NMR can also be increased using hyperpolarization,
and/or using two-dimensional, three-dimensional and higher-dimensional techniques.

Key Components of NMR:

 Magnet: Generates a strong, uniform magnetic field.

 RF Generator: Produces the RF pulses.

 Detector: Captures emitted RF signals as nuclei relax.

Chemical Shift:

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  The NMR signal's position depends on the local electronic environment, expressed in
ppm.

 Helps identify functional groups and bonding.

Spin-Spin Coupling:

 Interaction between neighbouring nuclei splits signals into multiplets.

 Provides information about molecular connectivity.

Applications:

 Structural determination of organic/inorganic compounds.

 Protein and nucleic acid structure elucidation.

 Quantitative analysis in chemistry and medicine (e.g., MRI).

Common NMR Types:

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 H-NMR: Focuses on hydrogen nuclei.

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 3C-NMR: For carbon nuclei.

 2D-NMR: Correlation spectra (e.g., COSY, NOESY).

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